Project Summary: Ion channel clustering in myelinated axons is essential for proper nervous system function. Ion channels are clustered at nodes of Ranvier through neuron-glia interactions. However, the mechanisms responsible for channel clustering and neuron-glia interactions remain poorly understood. Our work shows that multiple glia-dependent mechanisms contribute to node formation. Furthermore, these mechanisms converge on axonal and glial ankyrin and spectrin cytoskeletons. In this project, we will determine the importance of nodal spectrin cytoskeletons by analyzing conditional knockout mice lacking ?I and ?IV spectrins, singly and in combination. We also discovered that AnkyrinR (AnkR) can function as secondary Na+ channel clustering mechanism. However, the primary functions of AnkR in the nervous system are unknown. We generated a conditional allele for AnkR and will use this in loss-of-function studies to determine the role of AnkR in various cells throughout the nervous system. We performed differential proteomics on WT and AnkR-deficient brains together with immunoprecipitation to identify AnkR interacting proteins. We will investigate these interactions to further define the functions of AnkR in the nervous system. Finally, in a discovery aim, we will use a newly generated mouse (Nfasc-BioID) to perform in vivo proximity biotinylation at nodes and paranodes. We will then capture these proteins using streptavidin affinity purification, followed by mass spectrometry to identify biotinylated proteins. This will begin to uncover the node and paranode interactomes. We will validate and investigate these biotinylated proteins through localization, and gain and loss of function studies both in vitro and in vivo.